A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

Antigen-encoding bone marrow terminates islet-directed memory CD8+ T-cell responses to alleviate islet transplant rejection

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease, and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by conventional therapies, memory T cell-mediated attack is a substantial challenge in islet transplantation, and this will extend to application of personalized approaches using stem cell-derived replacement -cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement -cells. Here, we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions, inactivates established memory CD8(+) T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8(+) memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigen-specific memory T-cell responses, and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and, importantly, the clinical application of personalized -cell replacement therapies using patient-derived stem cells

Desmoglein-2 is important for islet function and β-cell survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes